This invention claims priority of a German filed patent application DE-A-199 14 049.9.
The invention concerns a microscope, preferably a confocal laser scanning microscope, having a light source, a detector, and two objectives, one of the objectives being arranged on each of the two sides of the specimen plane and the objectives being directed toward one another and having a common focus, and at least one beam splitter for distributing the illuminating light to the objectives, and a beam recombiner for combining the detected light coming from the objectives, being provided in the illumination/detection beam path.
Microscopes of the generic type, in particular microscopes in which two objectives that are directed toward one another and have a common focus in the image plane are provided, have been known in practical use for some time. Reference is made in this context, merely by way of example, to EP 0 491 289 B1. This document discloses a double confocal scanning microscope having the generic features. Specifically, what is described therein is a scanning microscope in which a non-polarizing beam splitter apparatus is provided to split the illuminating light into coherent portions. The beam splitter apparatus serves to illuminate the objectives directed toward one another, and to combine mutually coherent light beams from the objectives that are directed toward one another. High resolution can be obtained with the optical components implemented therein.
The microscope known from EP 0 491 289 B1 is a so-called xe2x80x9chigh-endxe2x80x9d microscope, in which an interferometric beam path is implemented. A microscope of this kind is of extremely complex design and is thereforexe2x80x94even in its basic versionxe2x80x94expensive compared to conventional microscopes. In addition, these xe2x80x9chigh-endxe2x80x9d microscopes are special constructions that require a great deal of room on optical benches and accordingly are also very susceptible to external influences. Above all, however, the xe2x80x9chigh-endxe2x80x9d microscopy provided for therein requires a special microscope, with no possibility of using conventional microscopes with their capabilities.
It is therefore the object of the present invention to be able to implement the ultrahigh-resolution microscope technique known from the generic existing art in conventional microscopes, for example in confocal laser scanning microscopes, in particular also by retrofitting.
The aforesaid object is achieved by a microscope which comprises at least one light source, at least one detector, and two objectives, one of the objectives being arranged on each of the two sides of the specimen plane and the objectives being directed toward one another and having a common focus; at least one beam splitter for distributing the illuminating light to the objectives, and a beam recombiner for combining the detected light coming from the objectives, being provided in the illumination/detection beam path; a modular assembly for grouping the objectives and the beam splitter/beam recombiner therein and an interface is provided with the modular assembly for connection to the illumination/detection beam path of the microscope.
In addition, the above object is achieved by a confocal laser scanning microscope comprising at least one detector, and two objectives, one of the objectives being arranged on each of the two sides of the specimen plane and the objectives being directed toward one another and having a common focus; at least one beam splitter for distributing the illuminating light to the objectives, and a beam recombiner for combining the detected light coming from the objectives, being provided in the illumination/detection beam path; a modular assembly for grouping the objectives and the beam splitter/beam recombiner therein and an interface is provided with the modular assembly for connection to the illumination/detection beam path of the microscope.
According to the present invention, it has firstly been recognized that conventional microscopes, for example confocal laser scanning microscopes, can be equipped at a later time with ultrahigh-resolution microscope techniques without thereby sacrificing the actual nature of the conventional microscope. It has furthermore been recognized that ultrahigh-resolution microscope techniques can be implemented in conventional microscopes even after the fact, specifically by the fact that the essential components that are responsible for the ultrahigh-resolution microscope technique are grouped into a retrofittable assembly. According to the present invention, therefore, the objectives and the beam splitter/beam recombiner are grouped into a modular assembly. This assembly has an interface for connection to the illumination/detection beam path of the microscope. Considered in and of itself, the modular assembly can be handled independently and can be connected with its interface to the microscope, a connection to the illumination/detection beam path being indispensable.
Advantageously, the assembly can be introduced with its interface into the microscope stand in place of a conventional objective or objective nosepiece, and thereby connected into the microscope (into its illumination/detection beam path). Conversion can thus be accomplished in simple fashion, namely after removing the objective or objective nosepiece, thereby making possible connection of the high-end assembly under discussion here.
In terms of a concrete embodiment of the assembly comprising the optical components, the components of the assembly are mounted on a baseplate. To prevent any temperature-related change in the beam path, it is particularly advantageous if the baseplate is manufactured from a material having a low coefficient of thermal expansion. Materials such as Invar or Super-Invar are thus suitable. These materials have almost no thermal expansion over the range of temperatures occurring here, so that any temperature-related change in the beam path or any corresponding misalignment can be almost ruled out.
In terms of particular secure positioning of the optical assembly, it is further advantageous if the optical components of the assemblyxe2x80x94preferably arranged on the baseplatexe2x80x94are arranged in a housing. To eliminate external influences, the housing could be hermetically sealed. Thermal insulation is also advantageous, specifically in order effectively to eliminate external temperature influences on the alignment.
In order effectively to prevent any misalignment, it would be possible to define inside the housing a defined temperature in whose vicinity the beam is aligned. In order to maintain this temperature, the optical assembly could comprise a component having a defined and at least largely constant heat emission. The heat-emitting component would need to be dimensioned so that it is suitable for keeping the assembly at a constant operating temperature, specifically in consideration of the thermal insulation possibly implemented therein. The heat-emitting component could be a laser light source, preferably a diode laser.
A further possibility for eliminating any misalignment due to thermal expansion is that the optical components of the assembly are designed, and are installed and arranged on the baseplate (optionally by way of special holders), in such a way that temperature-related expansions compensate for one another and therefore have no effect on the optical alignment state of the assembly. Very particularly, this can be implemented if individual components are designed such that they exhibit exclusively a linear expansion, i.e. an expansion in one direction. By way of corresponding end-located mounts and expansion characteristics in opposite directions from one another, it is possible for mutual compensation to occur as the temperature changes, with no change in the beam path. This action, too, at least largely prevents any misalignment.
The optical assembly could have further optical components in addition to the two objectives directed oppositely to one another and the beam splitter/beam recombiner. A further component that advantageously is also an integral constituent of the assembly is the specimen stage that is arranged between the objectives. When the assembly or the housing is connected to the microscope, the specimen stage is oriented horizontally so that the specimens to be examined can be placed in conventional fashion on the stage.
It is furthermore advantageous if the specimen stage is accessible from outside the housing. The specimen stage can thus be operated from outside, in particular can be loaded with the specimen to be examined, with no need to open the housing for that purpose. In the context of such an arrangement, the specimen stage and the specimen lying thereupon are protected by the housing.
The specimen stage could not only be loaded with a specimen from outside the housing, but also could be adjustable or displaceable from outside the housing. The specimen stage could be motor-driven for that purpose; an actuation member, preferably in the form of a joystick or trackball, can be provided for actuation of the specimen stage.
The assembly containing the optical components could furthermore comprise an optical system for displacing the pupil of the objectives used in the assembly. This makes possible adaptation to the beam path that has now been enlarged by the assembly. The optical system for displacing the pupil could be implemented by way of a virtual image or a real intermediate image. Concretely, the displacement of the pupil could be implemented by way of an optical system arranged in the illumination/detection beam path close to the coupling-in point. It is also conceivable for displacement of the pupil to be implemented by replacing the tube lens of the microscope. What is essential in this context is that a displacement of the pupil for adaptation purposes following introduction of the assembly into the microscope stand in place of the objective nosepiece is possible, specifically an adaptation of the position of the pupil of the objectives, since the objectives in the assembly, for example in an interferometer module, are farther away from the microscope stand than is the case in the conventional operating mode with objective nosepieces. Replacement of the tube lens, which could be arranged on a suitable lens wheel, is conceivable in principle.
The assembly could moreover comprise a mirror, arranged in the illumination/detection beam path, for deflecting the illumination/deflection beam. A compact design for the assembly can be achieved by using a mirror of this kind and by way of the resulting deflection of the beam.
A shutter could be arranged in the illumination/detection beam path between the coupling-in point and the beam splitter, preferably between the coupling-in point and the aforementioned mirror, specifically in order to block the beam or at least a partial beam.
A mirror could then once again be provided directly in front of the two objectives in order to deflect the illumination/detected light. These two mirrors then also serve to bring about a compact design for the entire assembly, allowing a reduction in the size of the housing required. A shutter can then once again be provided in the illumination/detection beam path between the beam splitter and the mirror, serving again to block or partially block the light beam in accordance with the requirements of the specific application.
It is furthermore conceivable for the assembly to comprise optical components in order to influence interference phenomena, in particular for phase compensation, specifically in order to implement an interferometric beam path. Very particularly, interferometers could be integrated for this purpose, for example Sagnac, Michelson, Twyman-Green, or Mach-Zehnder interferometers that are very particularly suitable for creating an interferometric beam path. The assembly could also comprise optical components for implementing 4Pi, SWFM,I2M, I3M, or I5M microscope technology, these being extremely complex xe2x80x9chigh endxe2x80x9d microscope technologies using a wide variety of microscope methods. For all these methods, it is essential to work with objectives directed toward one another.
In further advantageous fashion, the essential optical components of the assembly can be positioned for alignment purposes, specifically by way of actuators associated with the components. These actuators could be actuable via control units; the control units could be provided outside the assembly or inside the assembly (integrated thereinto). Particularly advantageously, the actuators are computer-controlled so that alignment can be accomplished by way of a suitable computer program. Corresponding user guidance for alignment purposes is conceivable.
Various parameters of the operating state of the assembly could moreover be detectable by way of detectors, the detectors being integral constituents of the assembly. These can be mechanical, electronic, and/or optical sensors that are particularly suitable for detection of the respective parameters for the purpose of ascertaining the operating state of the assembly. Automatic alignment of the system or the module is thereby possible.
As already mentioned above, the assembly could comprise its own light source, this light source being capable simultaneously of providing temperature control of the assembly. This light source could furthermore serve to generate an auxiliary beam for alignment of the components. The auxiliary beam could, in this context, be generated as an interferometric beam, the light source being an integral constituent of the assembly. It is also conceivable to couple the light source into the assembly as an external light source, any desired light source regardless of size being possible in this context. Coupling-in by way of optical fibers is conceivable.
The light source could be embodied as a solid-state laser, diode laser, or gas laser.
Lastly, be it noted that the assembly could comprise a fluorescent incident-light unit, a binocular tube, and/or an interface to a confocal unit. Further embodiments utilizing conventional microscope techniques are conceivable.